Methods: A cohort of 5,546 BRCA1 and 2,865 BRCA2 mutation carriers was used to evaluate risk of breast cancer associated with BARD1 Cys557Ser. In a second nonindependent cohort of 1,537 of BRCA1 and 839 BRCA2 mutation carriers, BARD1 haplotypes were also evaluated.

Results: The BARD1 Cys557Ser variant was not significantly associated with risk of breast cancer from single SNP analysis, with a pooled effect estimate of 0.90 (95% CI: 0.71–1.15) in BRCA1 carriers and 0.87 (95% CI: 0.59–1.29) in BRCA2 carriers. Further analysis of haplotypes at BARD1 also revealed no evidence that additional common genetic variation not captured by Cys557Ser was associated with breast cancer risk.

Conclusion: Evidence to date does not support a role for BARD1 variation, including the Cy557Ser variant, as a modifier of risk in BRCA1/2 mutation carriers.

Introduction

There is substantial interindividual variability in age at cancer diagnosis in BRCA1 and BRCA2 mutation carriers, which persists even among relatives that carry the same BRCA1 and BRCA2 mutation (1). Variation in genes that interact with BRCA1 and BRCA2 in the recognition and repair of DNA damage are strong candidates for study as genetic modifiers of BRCA1 and BRCA2 cancer risk. The BRCA1–BARD1 heterodimer is known to be important for BRCA1 function, with interaction mediated through the ring finger domains of the 2 proteins (2). In addition, although there is no evidence for a direct interaction between BARD1 and BRCA2, they do operate in the same DNA repair processes, exemplified by the fact that the BRCA2 partner RAD51, BARD1, and BRCA1 all relocate to proliferating cell nuclear antigen structures after irradiation (3).

The BARD1 Cys557Ser SNP (rs28997576) was first reported as a germ line alteration in a sporadic breast/uterine tumor (4). This variant lies between the ankyrin repeats and BRCT domains of BARD1, and the ectopically expressed Cys557 protein has growth suppression and proapoptotic effects relative to 557Ser (5). This SNP (minor allele frequency in Europeans: 0.025) has been reported to be associated with both breast cancer in the general population and familial breast cancer, but results have not shown consistent across all studies (6–12). Stacey and colleagues (6) initially reported that the Cys557Ser variant was associated with increased breast cancer risk in 756 Icelandic mutation carriers who carry BRCA2 999del5 founder mutation [OR = 3.1; 95% CI: 1.2–8.4]. However, subsequent studies reported no elevated risk in 228 Nordic BRCA1 and BRCA2 carriers (OR = 0.8, 95% CI: 0.3–2.0; ref. 8), or in 1,207 Polish BRCA1 mutation carriers (OR = 0.9, 95% CI: 0.4–2.2; ref. 10). There have been no previous haplotype-based studies assessing the role of BARD1 variation in breast cancer risk in BRCA1 and BRCA2 carriers specifically.

To resolve whether BARD1 is a modifier of BRCA1 and BRCA2-associated breast cancer risk, we undertook a large study to comprehensively assess the association of BARD1 Cys557Ser as well as haplotypic variation with cancer risk in BRCA1 and BRCA2 carriers.

Materials and Methods

Study sample

The design for this study has been described in detail previously (13). Briefly, eligible participants included adult women with documented disease-associated inherited mutations in BRCA1 or BRCA2. Mutations were included in the analysis if they were pathogenic according to generally recognized criteria (14, 15). Two overlapping cohorts of women with disease-associated BRCA1 and BRCA2 mutations were identitied (Table 1). First, a cohort of 5,546 BRCA1 and 2,865 BRCA2 mutation carriers from the multicenter CIMBA consortium (13) was used to evaluate risk of breast cancer associated with BARD1 Cys557Ser. Second, a cohort of 1,537 of BRCA1 and 839 BRCA2 mutation carriers participating in the MAGIC consortium was used to further explore the relationship between BARD1 haplotypes and breast cancer risk. Recruitment and genetic studies were approved by relevant ethics committees at all sites, and informed consent was obtained from each participant.

Laboratory methods

For analysis of the BARD1 Cys557Ser SNP, existing genotype data from BRCA1 and BRCA2 mutation carriers was requested from members of the CIMBA consortium. The primary methods used for genotyping were Sequenom iPlex (EMBRACE, -HEBON, kConFab, SWE-BRCA, PISA, Penn, Austria, Mayo, FCCC, GEMO, Georgetown, HEBCS) and by Taqman assays (OUH, Baylor, Beth Israel, City of Hope, Creighton, Dana Farber, NorthShore, IHCC, UCLA, University of Chicago, University of Texas Health Science Center, University of Utah, and Women's College Hospital; ref. 16) Genotypes for the INHERIT samples were typed by direct sequencing using an ABI Prism 3730xl DNA Analyser automated sequencer, with version 3.1 of the Big Dye fluorescent method according to the manufacturer's instructions (Applied Biosystems). Sequence data were analyzed using the Staden preGap4 and Gap4 programs. Samples from IHCC were typed by PCR-RFLP (10). SNP quality control measures included more than 95% success rate, Hardy–Weinberg Equilibrium P > 0.005. In addition, concordance of more than 98% for duplicate samples was required for studies that had included 2% duplicated samples for quality control purposes (all studies undergoing Sequenom iplex for BARD1 Cys557Ser, and all samples included in the haplotype substudy).

For studies of BARD1 haplotypic variation, 11 haplotype tag SNPs were identified and assayed at the University of Pennsylvania as previously described (16). The rs IDs were as follows: rs6712055, rs16852689, rs280621, rs13021937, rs13423596, rs10190829, rs6751923, rs4234006, rs28997576, rs3768708, rs1374230.

Statistical methods

To assess the relationship between BARD1 SNPs and breast cancer risk, proportional hazards models were used as previously described (16, 17). Briefly, participants were followed from the time of genetic testing or study ascertainment until the first diagnosis of breast cancer (the primary event in this analysis) or were censored at ovarian cancer. Participants who developed breast cancer were censored at bilateral prophylactic mastectomy if it occurred more than a year prior to the cancer diagnosis. This is to avoid censoring at bilateral mastectomies at which occult tumors were detected, but ages are rounded. The remaining participants were censored at the age at last observation. To address the problem of nonrandom sampling of mutation carriers with respect to the disease phenotype, analyses used the weighted Cox regression approach (17), where affected and unaffected individuals were differentially weighted such that observed breast cancer incidence rates in the study sample are consistent with established breast cancer risk estimates for BRCA1 and BRCA2 mutation carriers (18). Analyses assessing the association of the BARD1 Cys557Ser SNP combined heterozygote and homozygote variant carriers under a dominant model because of the rare frequency of this variant. Analyses were assessed separately for BRCA1 and BRCA2 mutation carriers, adjusted for Study group, ethnicity (non-Jewish Caucasian, Jewish or other), and year of birth cohort (decade of birth, categorized as <1940, 1940–1949, 1950–1959, 1960–1969, 1970–1989). There were 3,047 breast cancer events of 5,546 total for BRCA1 (55%) and 1,578 breast cancer events of 2,865 total for BRCA2 (55%) for the Cys557Ser censored analysis datasets. The remainders were censored for analysis. Secondary analyses adjusted for prophylactic oophorectomy, or assessed risk for the subset of carriers with mutations determined to result in unstable transcripts/proteins (class 1 loss of function mutations). R version 2.7.0 was used for single SNP statistical analyses.

To investigate haplotype effects, the Estimation-maximization algorithm (19, 20) was used to estimate haplotype frequencies as implemented in R version 2.1.1 subroutine haplo.em (21) as previously described (16). In this analysis, we included 607 breast cancer cases and 863 censored observations for BRCA1, and 813 breast cancer cases and 423 controls for BRCA2.

Results and Discussion

The frequency of the Cys557Ser SNP in the combined dataset (Table 1) was similar to published reports, with 4.4% of individuals carrying at least 1 rare allele (4.5% in BRCA1 carriers, 4.2% in BRCA2 carriers). There were no significant associations of Cys557Ser and breast cancer risk for carriers of BRCA1 mutations (HR = 0.90, 95% CI: 0.71–1.15) or BRCA2 mutations (HR = 0.87, 95% CI: 0.59–1.29). There was no evidence for heterogeneity by center for either BRCA1 or BRCA2 analyses (P > 0.5). There was also no evidence for association with additional adjustment for prophylactic oophorectomy, or when analyses were restricted to Class 1 mutations. For example, the HR for the subset of 3,882 individuals with BRCA1 Class 1 mutations was 0.84 (0.62–1.15), and for the 2,668 individuals with BRCA2 class 1 mutations was 0.96 (0.64–1.45).

For the haplotype analysis (Table 2), we also observed no overall effect of variation at BARD1 in either BRCA1 false discovery rate (FDR-corrected value of P = 0.152) or BRCA2 (FDR-corrected value of P = 0.134). No single BARD1 haplotype was significantly associated with breast cancer risk. Cys557Ser is represented by SNP 16 in Table 2 (BRCA1 haplotype 8 and BRCA2 haplotype 10). Since this variant was relatively rare (approximately 2% in both BRCA1 and BRCA2 carriers), estimates of its effect were not made in our primary analysis. When we fit a model that allowed the estimation of effects for haplotypes with at least 1% frequency in controls, no single haplotype was significantly associated with risk. The haplotype that contained the 557Ser allele was also not significantly associated with risk in either BRCA1 (HR = 0.91, 95% CI: 0.45–1.85) or BRCA2 (HR = 0.69, 95% CI: 0.28–1.72). Indeed, neither of these estimates was associated with increased risk of breast cancer as previously reported.

Analysis of BARD1 haplotype data: failure time analyses stratified on mutation using the MAGIC consortium data
a

The data presented here do not provide evidence that neither the BARD1 Cys557Ser SNP nor additional haplotypic variability not captured by Cys557Ser is associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers. Our sample size had more than 99% power to detect the effect size reported by Stacey and colleagues (6) of OR = 3.1. The study had more than 80% power to detect risk ratios of 0.89 (or 1.13) for BRCA1 carriers and 0.86 (or 1.17) for BRCA2 carriers. The upper 95% confidence limits on the rate ratio in our analysis exclude any substantial risk.

Conclusion

Our study found no evidence to support substantial associations of BARD1 variation with increased breast cancer risk in BRCA1 and BRCA2 carriers.

Austria MUV: The authors thank Daniela Muhr, Christine Fuerhauser-Rappaport, all the Hereditary Breast and Ovarian Cancer counselling clinics in Austria, and the many families who contributed to this study. This study was supported by the MUV Comprensive Cancer Center (Cluster Genetics and Epigenetics) and by the Austrian Society for Endocrinological Oncology.

HEBCS: HEBCS study was supported by Helsinki University Central Hospital Research Fund, Academy of Finland (132473), the Finnish Cancer Society and the Sigrid Juselius Foundation. The authors thank Drs. Kristiina Aittomäki, Carl Blomqvist, and Kirsimari Aaltonen for their help with patient data and samples.

IHCC: Support was provided by grant PBZ_KBN_122/P05/2004.

INHERIT: The authors thank Dr. Martine Dumont for sample management, Martine Tranchant for skillful technical assistance, and Dr. Frédéric Guénard for genotyping. They also thank Dr. Jacques Simard, Director of the INHERIT BRCAs research program, which is supported by the Canadian Institutes of Health Research (CIHR). This work was also supported by the Fonds de la Recherche en Santé du Québec (FRSQ)/Réseau de Médecine Génétique Appliquée (RMGA), the CURE Foundation, and the Canadian Breast Cancer Research Alliance (CBCRA). F. Durocher is a recipient of a chercheur-boursier from the Fonds de la Recherche en Santé du Québec (FRSQ) and a Research Career Award in the Health Sciences from CIHR/R&D Health Research Foundation.

kConFab: The authors thank Heather Thorne, Eveline Niedermayr, all the kConFab research nurses and staff, the heads and staff of the Family Cancer Clinics, and the Clinical Follow Up Study (funded by NHMRC grants 145684, 288704, and 454508) for their contributions to this resource, and the many families who contribute to kConFab. kConFab is supported by grants from the National Breast Cancer Foundation, the National Health and Medical Research Council (NHMRC) and by the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia. A.B. Spurdle and G. Chenevix-Trench are supported by a NHMRC Senior Research and Principal Research Fellowships, respectively.

Mayo: This research was supported by NIH grant CA128978, and NCI breast cancer specialized program of research excellence (SPORE) CA116201, and grants from the breast Cancer Research Foundation and the Komen Foundation for the cure. The authors thank Zachary Fredericksen, Robert Tarrell, and Vernon S. Pankratz for their assistance.

OUH: Thomas Sydenham, Anne-Marie Gerdes, and Torben A. Kruse are thanked for their contribution to this project.

Penn: This work was supported by HHSN21620074400C (to SMD), the Breast Cancer Research Foundation (to KLN), and R01-CA102776 and R01-CA083855 (to T.R. Rebbeck).

PISA: The authors thank the “Fondazione Cassa di Risparmio di Pisa” for a grant to MAC. All the surgeons (Dr. M. Roncella; Dr. E. Rossetti) and clinicians (Dr. A. Cilotto, C. Marini) who allowed us the identification of patients.

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